Bacteriocins from Streptococcus thermophilus

ABSTRACT

The invention relates to two new Streptococcus thermophilus bacteriocins having the amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 2, the signal peptides of these two bacteriocins, the nucleotide sequences encoding these bacteriocins especially an operon encoding the bacteriocins having the sequence SEQ ID NO: 3, the strains producing at least one of these bacteriocins especially the strain CNCM I-1351, a method for producing a supernatant extract comprising at least one of these two bacteriocins, and use of these bacteriocins in the preparation of food products, especially cheeses and acidified milks, and cosmetic products as active agent against pathogens.

TECHNICAL FIELD

The subject of the present invention is two bacteriocins fromStreptococcus (S.) thermophilus, a strain of S. thermophilus whichproduces these bacteriocins, a process for producing these bacteriocinsfrom this strain, as well as uses of these bacteriocins and/or of thisstrain in the preparation of food products or cosmetic products.

STATE OF THE ART

A bacteriocin is an antibacterial substance or an agent which is activeagainst bacteria comprising a protein portion which is involved in theantibacterial effect or antibiotic effect. A bacteriocin generally has anarrow activity spectrum or inhibition spectrum often limited to speciesclose to the species of the bacterium which produces it.

Currently, four S. thermophilus bacteriocins are known.

The first has, in particular, a molecular weight of 10 to 20 kD,exhibits thermolability at 90° C. and sensitivity to pepsin (Smaczny etal., Deutsche Molkerei-Zeitung, 105: 15, 460-464, 1984).

The second, which is described mainly by its bacteria inhibitionspectrum in EP 443543, has especially the capacity to inhibit the growthof bacteria of the genus Staphylococcus and Pseudomonas, and theinability to inhibit the growth of bacteria of the genus Lactococcus andEnterococcus, and of the species Bacillus cereus.

The third, which is described by Pulusani et al. (J. of Food Science,44:2, 575-578, 1979), strongly inhibits Pseudomonas, is not sensitive topepsin, and contains sugar residues.

Finally, the fourth, described by Gilano et al.(Microbiologie-Aliment-Nutrition, 8, 21-30, 1990), is not sensitive topepsin, contains sugar residues and does not pass across a membrane witha porosity of 100 kD.

Now, S. thermophilus is of major importance in the food sector, beingespecially involved in the preparation of dairy products such as yogurtsand some cheeses for example. Furthermore, very few bacteriocins existwhich are active at the same time against Bacillus, Clostridium andListeria. It may therefore be more useful, in other words there is aneed for a broader range of bacteriocins produced by representatives ofthis species in order to have especially a broader antibacterialactivity spectrum, in particular in the context of this type of product.

The aim of the present invention is to respond to this need.

SUMMARY OF THE INVENTION

One of the subjects of the present invention is the characterization ofthe amino acid sequence of two new S. thermophilus bacteriocins, as wellas their signal peptide which permits their excretion.

The nucleotide sequences encoding these two bacteriocins are alsoanother subject of the invention.

The Streptococcus thermophilus strains producing at least one of thebacteriocins according to the invention are also another subject of theinvention, especially the strain CNCM I-1351 of S. thermophilusdescribed below, which is capable of producing the two bacteriocinsaccording to the invention.

The process for producing an extract of at least one bacteriocinaccording to the present invention is also another subject of thepresent invention.

Finally, the last subject of the present invention is the use of thebacteriocins according to the invention and the use of their nucleicsequence, as well as their signal sequence.

DETAILED DESCRIPTION OF THE INVENTION

A strain CNCM I-1351 of S. thermophilus was isolated from a fermenteddairy product from Czechoslovakia and it was observed, surprisingly,that it has the remarkable property of inhibiting the growth of a broadrange of bacteria. This strain was deposited on May 8, 1993, accordingto the Budapest Treaty, at the Collection Nationale de Cultures deMicroorganismes, PASTEUR INSTITUTE, 25, Rue du Docteur Roux, F-75724PARIS CEDEX 15, France where it was thus assigned the No. I-1351. Anyand all restrictions on the availability to the public of the materialso deposited will be irrevocably removed upon the granting of a patentfor the present invention.

Details on this strain relating especially to its morphology, thefermentation of sugars and the like are given below:

Morphology:

Non-flagellated chain-forming cocci. No formation of spores.

Gram-positive microorganisms, catalase negative and facultativeanaerobes.

Fermentation of sugars:

Production of lactic acid from D-glucose, lactose, sucrose, raffinose.No production of lactic acid from mannose, fructose, galactose.

Others:

Strain producing at least two bacteriocins, one protein for immunity tobacteriocins, and exopolysaccharides having texturizing properties.

The culture supernatant of the CNCM I-1351 strain therefore has arelatively broad anti-bacteria activity spectrum. Among the bacteriasensitive to this supernatant, there may be included Streptococcusthermophilus, Lactococcus lactis, Lactococcus lactis biovardiacetylactis, Lactococcus cremoris, Enterococcus faecalis, Enterococcusfaecium, Lactobacillus fermentum, Lactobacillus helveticus,Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillusbrevis, Leuconostoc cremoris, Leuconostoc mesenteroides, Bifidobacteriumbreve, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacteriuminfantis, Propionibacterium, Listeria innocua, Listeria momocytogenes,Micrococcus varians, and the spores and the vegetative cells ofClostridium botulinum, Clostridium tyrobutyricum, Clostridiumbifermentans, Clostridium sporogenes, Bacillus subtilis, Bacilluspumilus and Bacillus cereus, for example (Bacteries lactiques, vol 1,1994, Lorica edition).

It was then possible to isolate from this CNCM I-1351 strain two proteinfactors called bacteriocins, which are responsible for thisantibacterial activity.

To this end, the first bacteriocin according to the invention which isnamed in this disclosure "thermophilin 1", has the sequence SEQ ID NO: 1described in the sequence listing below.

Furthermore, it is possible to envisage that this bacteriocin may havean antibacterial activity having a broader or more specific spectrum forone genus or one bacterial species than that exhibited by thermophilin1, when the latter has a sequence differing from the sequence SEQ ID NO:1 in a substitution, a deletion and/or an insertion of at least oneamino acid for example. Indeed, it is already known from EP 521240 thatnisin Z has a more advantageous activity spectrum than nisin A, whereasit differs from nisin A only in a substitution of an amino acid.

That is why all the bacteriocins having a substitution, a deletionand/or an insertion of at least one amino acid in their originalsequence SEQ ID NO: 1 can be considered as bacteriocins according to thepresent invention.

The second bacteriocin according to the invention, called in thisdisclosure "thermophilin 2", has the sequence SEQ ID NO: 2 described inthe sequence listing below. It is also possible to envisage that thisbacteriocin may have an antibacterial activity when it has a sequencewhich differs from the sequence SEQ ID NO: 2 by a substitution, adeletion and/or an insertion of at least one amino acid for example. Tothis end, all bacteriocins having at least one of the modificationsdescribed above in their original sequence SEQ ID NO: 2 can beconsidered as bacteriocins according to the present invention.

In addition, the nucleotide sequences encoding thermophilin 1 andthermophilin 2 are also another subject of the present invention becausethey can each be used to confer, by transformation, on bacteria, yeastor plants for example, a capacity to inhibit certain bacteria. Thesenucleotide sequences can thus be relatively variable because of thedegeneracy of the genetic code, and may especially be comprised withinan operon of the SNCM I-1351 strain having the nucleic sequence SEQ IDNO: 3 described in the sequence listing below.

In particular, it is possible to use the nucleic sequence comprisingnucleotides 221 to 475 of the sequence SEQ ID NO: 3, which encodesthermophilin 1 with its signal peptide. However, it is more advantageousto use only the sequence encoding the signal peptide, from nucleotide221 to 288 of the sequence SEQ ID NO: 3, to fuse it to a gene ofinterest, so as to be able to permit the excretion, by a Streptococcusthermophilus strain, of the protein encoded by this gene of interest.Likewise, it is more advantageous to use only the sequence encoding theexcreted thermophilin 1, from nucleotide 289 to 475 of the sequence SEQID NO: 3, in order to be able to fuse it to a signal sequence in anexpression plasmid, for example, so as to express it in a microorganismother than S. thermophilus.

Likewise, the nucleic sequence comprising nucleotides 495 to 686 of thesequence SEQ ID NO: 3, which encodes thermophilin 2 with its signalpeptide, can be used. However, it is more advantageous to use only thesequence encoding the signal peptide, from nucleotide 495 to 557 of thesequence SEQ ID NO: 3, in order to be able to permit the excretion, by aStreptococcus thermophilus strain, of any protein fused to this peptide.Likewise, it is more advantageous to use only the sequence encoding theexcreted thermophilin 2, from nucleotide 558 to 686 of the sequence SEQID NO: 3, in order to be able to fuse it to a signal sequence in anexpression plasmid, for example, in order to express it in amicroorganism other than S. thermophilus.

Finally, as it has been observed that certain S. thermophilus strains,other than the strain CNCM I-1351, exhibit an inhibition spectrumsimilar to that exhibited by the strain CNCM I-1351 and a resistance tothe latter (especially the strains Sfi12 and 25 described below), it ishighly probable that these strains can produce at least one of thebacteriocins of the present invention, at the same time as an immunityprotein conferring this resistance. To this end, all the strains capableof producing at least one of the bacteriocins described above areincluded in the present invention.

In the process for producing an extract comprising at least onebacteriocin according to the present invention, an S. thermophilusstrain which produces at least one of the bacteriocins is cultured in amedium and under conditions favorable to the growth of S. thermophilusuntil the medium contains 10⁷ -10⁹ microorganisms of the strain per ml,the culture obtained is centrifuged and then an extract of thesupernatant comprising at least one of the bacteriocins is prepared.

To produce this extract, the S. thermophilus strain producing at leastone of the bacteriocins according to the present invention, especiallythe strain CNCM I-1351 of S. thermophilus, can therefore be cultured ina medium and under conditions favorable to the growth of S.thermophilus. It can be cultured especially in an MSK medium (skimmedcow's milk supplemented with yeast extract) or in a HJ medium (cow'smilk ultrafiltration permeate supplemented with yeast extract andsoytone) for example. It is preferably cultured in a medium which isselective for Streptococcus, such as the M 17 medium described by P. E.Terzaghi et al., J. Appl. Microbiol., 29, 807-813 (1975), supplementedwith 0.5-2% of a sugar which can be fermented by S. thermophilus,especially sucrose, lactose or glucose for example.

Such a medium can be prepared by mixing 95 ml of a basic medium and 5 mlof a solution containing 10 g of fermentable sugar per 100 ml of water,the solution of fermentable sugar and the basic medium having each beensterilized separately at 121° C. for 15 min and the basic medium havingbeen prepared by dissolving the following components in 950 ml ofboiling water:

    ______________________________________                                        trypsin casein hydrolysate                                                                             2.5 g                                                pepsin meat hydrolysate  2.5 g                                                papain soya bean hydrolysate                                                                           5.0 g                                                yeast extract            2.5 g                                                meat extract             5.0 g                                                beta-glycerophosphate    19 g                                                 Mg sulphate              0.25 g                                               ascorbic acid            0.5 g                                                ______________________________________                                    

The strain can be cultured in the medium favourable for the growth of S.thermophilus at 37°-48° C., for 2-8 h for example, until the mediumcontains about 10⁷ -10⁹ microorganisms of the strain per ml, a value ofabout 10⁸ microorganisms/ml corresponding, on the one hand, to anoptical density of the medium, measured at 600 nm (OD₆₀₀), of about 3.6and, on the other hand, to the concentration reached in a cow's milk atthe point where it coagulates under the effect of the acidificationproduced by the cultured strain.

To prepare a crude extract of the supernatant, it is possible to use anyappropriate precipitation method such as precipitation withtrichloroacetic acid, "salting out" or solvent precipitation forexample. Preferably, in order to prepare this crude extract, the pH ofthe supernatant is adjusted to 1.0-2.0 with H₃ PO₄, a precipitate isremoved, and one or more successive precipitations are carried out withtrichloroacetic acid each followed with resuspension in an aqueoussuspension with trifluoroacetic acid.

Use of the bacteriocins and/or of a Streptococcus thermophilus strainwhich produces these bacteriocins according to the present invention isprovided for in the preparation of food products or cosmetic products.

A culture of the Streptococcus thermophilus strain can be used inparticular as starter in the preparation of cheeses, especially ofcheeses of the mozzarella type (to avoid the holes produced by Bacilluspolymixa whose spores survive the fermentation), of the Swiss type (suchas Gruyere or Emmental, to combat contamination by Clostridiumtyrobutyricum), of the vacherin type (to combat contamination byListeria monocytogenes), and of the "sere" type (French name for soft orcream cheese), or in the preparation of acidified milks, especially ofyogurt or of powdered milk for infant formulas, for example.

In particular, the Streptococcus thermophilus strain can be cultured inmilk in combination with a Lactobacillus bulgaricus strain which ismildly sensitive to thermophilin (for example the strain YL5 describedbelow), to avoid the post-acidification of the yogurt due to L.bulgaricus.

The bacteriocins, especially in the form of a crude or purified extract,or the strain can also be used as additive or active agent againstpathogenic bacteria, especially in the preparation of meat products suchas mousses, as active agent against the growth of clostridia spores,especially Clostridium botulinum, or in the preparation of creams orlotions, as active agent against pathogenic bacteria of the skin, oralternatively in the preparation of oral health products, as activeagent against pathogenic bacteria of the buccal cavity, especiallyagainst Streptococcus sobrinus, for example.

The bacteriocins according to the present invention are characterized ingreater detail below by means of various microbiological, biochemicaland genetic data illustrating their properties. The percentages aregiven by weight.

Unit of antibacterial activity--"Agar Well-Test"

Within the framework of the present disclosure, antibacterial activityis defined in terms of arbitrary units.

One arbitrary unit (au) is defined as the reciprocal of the rate of thehighest dilution at which a sample still exhibits antibacterial activityin the test known to persons skilled in the art under the name of "agarwell test", the English expression which literally means a test using awell cut out in agar.

A standard sample of a supernatant of an S. thermophilus cultureaccording to the present invention prepared under the standardconditions illustrated in Example 1, typically exhibits an activity of32 au for a volume of 70 μl. This therefore means an activity of 460au/ml.

A standard crude extract of bacteriocin, obtained from the culturesupernatant illustrated in Example 1 by clarification followed by twosuccessive precipitations with trichloroacetic acid each followed with aresuspension in aqueous suspension with trifluoroacetic acid, typicallyhas an activity of about 1.4×10⁵ au/ml.

It is with the aid of the agar well test that it is determined whether asample still has an antibacterial activity at a given dilution rate.

In order to do this, 35 ml of M 17 medium are poured into a Petri dishand 1% sucrose and 1.5% agar are added thereto.

5 ml of M 17 medium to which 1% sucrose and 0.75% agar are added areinoculated with 5 μl of a culture, prepared during the previous night,of a strain of S. thermophilus which is typically sensitive to thepresent bacteriocin (typical indicator), in this case the strain Sfi3for example.

The 5 ml are poured over the 35 ml and left to dry for 15 min under alaminar flow. Holes of 5 mm in diameter are punched in the culturemedium.

The test samples are poured into the holes, in an amount of 70 μl perhole. The incubation is carried out for 6 h under anaerobic conditionsat 42° C. During this incubation, the typical indicator strain has grownand inhibition halos are visible. The dilution rate at which a sample nolonger exhibits antibacterial activity is the dilution rate from whichan inhibition halo is no longer distinguished.

Inactivation by enzymes

With the aid of the agar well test, on agar inoculated with the typicalindicator strain as described above, it is determined whether thepresent bacteriocins are inactivated or not by various enzymes.

For all the enzymes used except for lipase, 1 μg/ml to 10 mg/ml ofenzyme is added to the standard crude extract diluted 33× in the bufferrecommended by the enzyme supplier, so as to obtain samples of 70 μl at300 au. The enzyme is then allowed to act for 30 min, at the temperaturerecommended by the supplier, before placing the whole in the well of theagar well test.

On the other hand, for the commercial lipase, 1 μl of a mixture ofinhibitors (1.25M EDTA; 0.25% pepstatin A (p4265 Sigma); 0.25% E-64(E3132 Sigma); 0.25% aprotinin (A1153 Sigma)) is first added to 100 μlof a solution comprising 200 μg/ml of lipase, the inhibitors are allowedto act for 45 min at room temperature, 5 μl (450 au) of diluted standardcrude extract are then added and allowed to react for 30 min at 37° C.,then 70 μl of the mixture are deposited in the well of the agar welltest. The buffers used for the dilutions are the following.

pH 2.0: 100 mM maleic acid adjusted with NaOH,

pH 7.0: 100 mM phosphate buffer (K₂ HPO₄ /KH₂ PO₄),

pH 7.5: 100 mM phosphate buffer (K₂ HPO₄ /KH₂ PO₄),

pH 7.75: 100 mM Tris-Cl.

The diameter of the inhibition halo is compared with the controldiameter of the halo obtained without addition of enzyme which, for eachbuffer and at each incubation temperature, is about 14 mm.

Table I below presents the results obtained with the tested enzymes. Inthis table, the enzyme is designated by its type, the name of thesupplier and the item number of the supplier. The inactivation of thebacteriocin is indicated as a function of the concentration of theenzyme added. The figure 0 means that there is no longer any halo, inother words that the antibacterial activity of the present bacteriocinwas impaired by the incubation with the enzyme. The figure 14 indicatesthat there is still a halo of 14 mm corresponding to the fullantibacterial activity of the present bacteriocin.

                                      TABLE I                                     __________________________________________________________________________                   Concentration                                                                        pH of the                                                                          Incubation                                                                            Inactivation                               Enzymes        (μg/ml)                                                                           buffer                                                                             temperature (°C.)                                                              (mm)                                       __________________________________________________________________________    Pepsin (SIGMA P-700)                                                                         10     2.0  37      0                                          Proteinase K (MERCK 1000 144)                                                                4      7.0  37      0                                          Ficin (SIGMA P-3266)                                                                         10     7.0  37      0                                          Pronase E (SIGMA P-8038)                                                                     10     7.5  37      0                                          Nagarse (SIGMA P-4789)                                                                       10     7.5  37      0                                          Trypsin (SIGMA T-8128)                                                                       10     7.5  25      0                                          α-chymotrypsin (SIGMA C-7762)                                                          1      7.75 25      0                                          Catalase (SIGMA C-10)                                                                        10000  7.75 25      14                                         α-amylase (SIGMA λ-0521)                                                        1      7.75 25      14                                         Lipase (SIGMA L-0382) +                                                                      200    7.75 37      14                                         protease inhibitors                                                           __________________________________________________________________________

All the proteases suppress the antibacterial activity of thesupernatant, which demonstrates that a protein portion is involved inthis activity.

The fact that no influence of catalase is observed on the antibacterialactivity of the bacteriocins also demonstrates that the inhibition ofthe growth of the typical indicator strain is not due to theantibacterial activity of H₂ O₂ which is known to have a similaractivity to that of bacteriocins, since H₂ O₂ would have been degradedby catalase.

Likewise, the fact that no inactivation of the antibacterial activity byα-amylase is observed demonstrates the absence of α-amylase-hydrolysablesugars involved in this antibacterial activity.

In addition, the fact that the lipase has no influence on theantibacterial activity also demonstrates the absence of a lipid fractioninvolved in this activity.

Inhibition spectrum

With the aid of the agar well test, on agar inoculated with variousstrains of spores or bacteria, it is determined whether the culturesupernatant of the CNCM I-1351 strain producing the two bacteriocinsaccording to the invention, has an inhibitory activity on the growth ofthese various bacteria, in other words, an inhibition spectrum isdetermined for this supernatant.

To do this, the inhibitory effect on the growth of the tested strainproduced by a sample of supernatant exhibiting an activity of 300 au atpH 7.0 is observed in relation to the effect, which is normally zero, ofthe same sample previously deactivated by incubation at 37° C. for 30min in the presence of 5 μg/ml of proteinase K.

To carry out these assays, FALCON 3046 Multiwell tissue culture platesare used. 6 ml of M17 medium containing, in addition, 1% lactose and1.5% agar (M17L medium) is covered with 700 μl of M17 medium containing,in addition, 1% lactose and 0.6% agar inoculated with 1% of a culture ofthe test strain prepared during the previous night and diluted to anOD₆₀₀ of 0.1.

When the test strain has to grow from spores, the inoculation is carriedout with 10⁵ -10⁶ spores per ml of covering medium.

When the test strain is not a Lactococcus, a Streptococcus or anEnterococcus, the M17L medium is replaced with a standard mediumfavourable to the growth of the bacterium in question, especially theMRS medium comprising, in addition, 2% glucose for Lactobacillus,Pediococcus, Leuconostoc and Bifidobacterium (Sanofi DiagnosticsPasteur, France), the RCM medium for the spores or vegetative cells ofClostridium (Oxoid, England), and the BHI medium (Difco, USA) for theother bacteria tested.

Two holes 5 mm in diameter and 5 mm deep are punched per plate. A 70 μlsample at 300 au of the present bacteriocin is placed in one of theholes, and in the other the same sample previously deactivated. Theincubation is carried out at a temperature favourable to the growth ofthe tested strain for a period necessary for it to cover the plate witha visible bacterial lawn.

The effect or the degree of inhibition is characterized by the diameterof the inhibition halo observed. It is considered that the inhibition isvery high (++++) if the halo has a diameter of 16-18 mm, high (+++) fora diameter of 11.5-15.5 mm, average (++) for a diameter of 7.5-11 mm,weak (+) for a diameter of 5-7.5 mm and zero (-) if no halo is observed.

More than 74 strains of lactic acid bacteria of various species andsubspecies are thus tested and it is observed that only about 7% of themare resistant to the supernatant. The details of the result of thesetests is presented in Table II below. In this Table II, as in thefollowing tables, the strain name or No. indicated is the No. which isattributed to it in the Nestle collection (address: NESTEC S. A.,Research Centre, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland).The temperature indicated is the incubation temperature during the test.

                  TABLE II                                                        ______________________________________                                        Species           No.      T (°C.)                                                                        Inhibition                                 ______________________________________                                        Streptococcus thermophilus                                                                      YS3      42      +++                                                          YS4      42      +++                                        (The strains Sfi 12 and 25                                                                      YS11     42      +++                                        exhibit resistance to the                                                                       YS7      42      +++                                        strain CNCM I-1351 and an                                                                       YS8      42      +++                                        anti-bacteria activity                                                                          YS20     42      +++                                        spectrum which is similar                                                                       Sfi3     42      +++                                        to this strain)   Sfil18   42      +++                                                          Sfi19    42      +++                                                          Sfi20    42      +++                                        (The strain STII exhibits                                                                       Sfi16    42      +++                                        resistance only to the                                                                          ST11     42      -                                          strain CNCM I-1351; it                                                                          Sfi12    42      -                                          appears however that less than                                                                  Sfi25    42      -                                          5% of streptococci are                                                        capable of expressing                                                         such a resistance)                                                            Lactococcus lactis                                                                              SL2      30      ++                                         (Nisine producers)                                                                              SL13     30      ++                                                           SL16     30      ++                                                           SL25     30      ++                                                           SL31     30      ++                                                           SL63     30      ++                                         Lactococcus lactis                                                                              SLP26    30      ++                                                           SLP29    30      ++                                                           SLP24    30      ++                                                           SL64     30      ++                                                           SL58     30      ++                                                           SL40     30      ++                                         Lactococcus lactis biovar                                                                       SD39     30      ++                                         diacerylactis     SD80     30      ++                                                           SD57     30      ++                                                           SD11     30      ++                                                           SD113    30      ++                                         Lactococcus cremoris                                                                            SC20     30      ++                                                           SC15     30      ++                                                           SC11     30      ++                                                           SC145    30      ++                                                           SC63     30      ++                                                           SC28     30      ++                                         Enterococcus faecalis                                                                           SFS1     30      +                                                            SFS2     30      +                                                            SFS10    30      +                                          Enterococcus faecium                                                                            SFM1     30      ++                                                           SFM3     30      ++                                                           SFM6     30      ++                                                           SFM10    30      ++                                                           SFM14    30      ++                                                           SFM9     30      +                                          Lactobacillus fermentum                                                                         L26      30      ++                                                           L50      30      ++                                                           L28      30      ++                                                           LF16     30      ++                                                           LF15     30      ++                                         Lactobacillus helveticus                                                                        LH91     40      ++++                                                         LH2      40      +++                                                          LH3      40      +++                                                          LH1      40      +++                                        Lactobacillus acidophilus                                                                       LQ1      40      ++                                                           LQ3      40      +                                                            LQ10     40      ++                                                           LQ21     40      +                                                            LQ23     40      ++++                                                         LQ26     40      -                                          Lactobacillus brevis                                                                            LB2      30      +++                                                          LB10     30      -                                                            LB13     30      +++                                        Lactobacillus bulgaricus                                                                        YL12     40      ++++                                                         YL2      40      +                                                            YL5      40      ++                                                           LB32     40      +++                                        Leuconostoc cremoris                                                                            LCC1     30      ++                                                           LCC7     30      ++                                                           LCC2     30      ++                                         Leuconostoc mesenteroides                                                                       LCM9     30      ++                                                           LCM10    30      ++                                                           LCM18    30      ++                                         ______________________________________                                    

In this Table II, it is observed that the inhibition spectrum of thesupernatant is narrow in the sense that for certain Lactobacillusspecies, such as Lactobacillus acidophilus, Lactobacillus brevis andLactobacillus bulgaricus, for example, the degree of inhibition isheterogenous. However, for other species such as L. fermentum, L.helveticus and Lactococcus for example, the degree of inhibition ishomogeneous.

This is advantageous in the light of the fact that it is very difficultto distinguish one strain from another within the same species. It istherefore possible to envisage an advantageous use of the supernatant orof the purified bacteriocins for distinguishing between industrialstrains.

It is also possible to envisage the use of a strain producing at leastone of the bacteriocins according to the present invention, in culturewith another lactic acid bacterium strain which is naturally resistant,or slightly sensitive, to the bacteriocin(s) produced in the medium.Yogurts, especially yogurts exhibiting reduced post-acidification, forexample, can thus be produced.

It is also observed that the supernatant inhibits the growth of the sixnisin-producing strains of L. lactis. This proves that the presentbacteriocin is not nisin. This is confirmed by the fact that the presentbacteriocin is inactivated by trypsin at 10 μg/ml (cf. Table I), whichis not the case for nisin.

However, the inhibition spectrum of the supernatant of a cultureproducing the two bacteriocins of the invention is also broad in thesense that it is not limited to species of lactic acid bacteria but thatit extends to other species of Gram-positive bacteria, especially to thefood bacteria Bifidobacterium, to the undesirable or pathogenic bacteriaPropionibacterium, Listeria innocua, Listeria monocytogenes andMicrococcus varians, and to the spores and cells of numerous pathogenicbacteria of the genus Clostridium and Bacillus, for example, asdemonstrated by the results presented in Table III below.

                  TABLE III                                                       ______________________________________                                        Species           No.       T (°C.)                                                                        Inhibition                                ______________________________________                                        Bifidobacterium breve                                                                           BBR27     37      +++                                                         BBR4      37      +++                                                         BBR39     37      +++                                       Bifidobacterium longum                                                                          BL20      37      +++                                                         BL18      37      +++                                                         BL22      37      +++                                       Bifidobacterium bifidum                                                                         BB7       37      +++                                                         BB9       37      +++                                                         BB12      37      +++                                       Bifidobacterium infantis                                                                        B16       37      +++                                                         B11       37      +++                                       Propionibacterium PP1       30      +++                                       Clostridium botulinum                                                                           CB1       30      ++                                        (Spores and vegetative                                                                          CB2       30      ++                                        cells)                                                                        Clostridium tyrobutiricum                                                                       107001    30      +                                         (Spores and vegetative                                                                          107002    30      ++                                        cells)                                                                        Mixture of spores of                                                          Clostridium sporogenes                                                                          100021                                                      Clostridium fermentum                                                                           100022    30      ++                                        Clostridium butilinum                                                                           A-69; B-213;                                                                  BKA40;                                                                        B-73-211;                                                   (6 strains)       A-80-                                                                         124clovis;                                                                    B-1-NCA                                                     Listeria innocua  24        30      +                                                           25        30      +                                                           27        30      +                                                           39        30      +                                                           40        30      +                                                           41        30      +                                         Listeria monocytogenes                                                                          57        30      ++                                                          58        30      ++                                                          59        30      ++                                                          60        30      ++                                                          61        30      ++                                                          62        30      ++                                        Bacillus subitilis                                                                              A2        30      ++                                        (spores and vegetative                                                                          A3        30      ++                                        cells)            A13       30      ++                                                          A14       30      ++                                                          A15       30      ++                                        Bacillus pumilus  B2        30      ++                                        (Spores and vegetative                                                        cells)                                                                        Bacillus cereus   C14       30      ++                                        (spores and vegetative                                                        cells)                                                                        Micrococcus varians                                                                             MCV1      30      ++                                        Micrococcus luteus                                                                              MCL1      30      -                                         (nisin indicator)                                                             ______________________________________                                    

The results illustrated in this Table III make it possible, inter alia,to envisage advantageous uses of this supernatant or of the purifiedbacteriocins, as additive in the preparation of food products as activeagent against pathogenic agents, especially in meat products againstClostridium, in cheeses against Listeria monocytogenes and C.tyrobutyricum, or in fresh pasta or sauces for fresh pasta againstBacillus from which the above strains indeed originate for example.

Finally, the present bacteriocins exert no inhibitory effect on thegrowth of Gram- bacteria, as can be observed in the light of the resultsillustrated in Table IV below.

                  TABLE IV                                                        ______________________________________                                        Species          No.      T (°C.)                                                                        Inhibition                                  ______________________________________                                        Escherichis coli BZ234    37      -                                           Salmonella thyphimurium                                                                        274      37      -                                                            273      37      -                                           Pseudomonas aeruginosa                                                                         5        37      -                                                            13       37      -                                           Pseudomonas fluorescens                                                                        11       37      -                                                            12       37      -                                           ______________________________________                                    

Heat resistance, stability

The bacteriocins present in the extract obtained under the conditionsillustrated in Example 1 do not exhibit good stability to preservationat 4° C. if the extract is not previously heated. On the other hand,they exhibit good stability to preservation if the extract is sharplyheated for at least 15 min at 90°-121° C. for example.

It was checked in particular that more than 50% of the activity of suchan extract is preserved after 5 months of preservation at 4° C. if thesaid extract was heated beforehand for 20 min at 94° C. on a water bathfor example. It was also checked that 100% of the activity is preservedafter heating the said extract for 60 min at 100° C. (test carried outon a thermostatted oil bath on 1 ml of the supernatant, concentrated orotherwise, of a culture of a strain of S. thermophilus according to thepresent process).

On the other hand, the present bacteriocins preserve only about a thirdof their activity after a sterilizing treatment of 30 min at 121° C.(test carried out on 40 ml of the non-concentrated supernatant of aculture of a strain of S. thermophilus according to the present process)for example.

Finally, by ultrafiltration tests on Amicon filters followed by gelelectrophoresis (SDS-PAGE), it is observed that the bacteriocins of thepresent invention in the supernatant of a culture of S. thermophilus,especially in the supernatant of the standard culture obtained inExample 1, exist in the form of aggregates of molecular weight (MW)greater than 10 kDa, of which 67% exhibit a MW of less than 100 kDa at33% exhibit a MW greater than 100 kDa.

Purification of the bacteriocins

In the description which follows, the percentages of trifluoroaceticacid and acetonitrile are given by volume.

1 litre of a culture of the CNCM I-1351 strain is produced in an M17medium supplemented with 1% sucrose, for 6 h, at 42° C. and underanaerobic conditions.

20 g of XAD-7 resin (Sigma) are then added directly to the culture andthe whole is stirred gently for 1 h at 4° C. The mixture is thenfiltered through a Schleicher & Schvell filter (Germany) No. 604, thenthe resin retained on the filter is washed with 1 litre of a 50 mMacetic acid solution pH 5.2, in order to remove the bacteria. The resinis then placed in a column and the bacteriocins are eluted with 45 ml ofa solution comprising 70% acetonitrile and 0.1% trifluoroacetic acid(TFA). An eluate comprising both bacteriocins is then obtained.

These two eluted bacteriocins are then separated in the followingmanner.

The volume of eluate is first reduced to 24 ml bycentrifugation/freeze-drying (Speedvac, Savant Instrument), the volumeobtained is then adjusted to a concentration of 2M NaCl and 250 mMTris.Cl, pH 8, to a volume of 50 ml, then this volume is injected into aPhenyl Superose HR 16/10 column with hydrophobic interaction (Pharmacia)previously equilibrated with a buffer comprising 50 mM Tris.Cl, pH 8 and2M NaCl. 200 ml of the preceding buffer, 100 ml of a linear gradientstarting with the preceding buffer and ending with a 50 mM Tris.Clsolution, pH 8, 100 ml of the preceding solution, 60 ml of pure water,60 ml of 50 mM Tris.Cl solution, pH 8, and finally 60 ml of pure waterare then passed successively at a rate of 4 ml/min.

50 μl of each fraction collected at the outlet of the column are thendiluted in 50 μl of 0.1% TFA, then the antibacterial activity of eachmixture is tested by the agar well test described above.

It is thus observed that the 470th to 490th ml fractions exhibit anantibacterial activity. These fractions are then mixed, the volume ofthis mixture is reduced by centrifugation/freeze-drying to 1 ml, thenthis reduced volume is injected into a Pep RPC HR 5/5 column (Pharmacia)previously equilibrated with a 0.1% TFA solution, called in thisdisclosure "solution A". A solution of elution "B" comprising 70%acetonitrile and 0.097% TFA is also prepared. 1 ml of solution A, 9 mlof a linear gradient starting with solution A and ending with a 50/50mixture of solutions A and B, 2 ml of this latter mixture, 7 ml of alinear gradient starting with this latter mixture and ending with asecond 20/80 mixture of solutions A and B, 2 ml of a linear gradientstarting with this second mixture and ending with solution B, then 2 mlof this latter solution are then successively passed through the columnat a rate of 1 ml/min.

The antibacterial activity of the fractions at the outlet of the columnis then determined by the agar well test as described above. All thefractions from the 14th to the 22nd ml exhibit an antibacterialactivity. On the other hand, two major protein peaks, observed at anoptical density of 215 mn, are distinguished in fractions 15 and 21 (inmillilitre).

Sequencing of the bacteriocins

The N-terminal part of the proteins contained in fractions 15, 18, 20,21 and 22 is sequenced using an Applied Biosystems 4774 automaticsequencer.

The presence of a peptide having a sequence of 48 amino acids which isidentical to that, for the N-terminal part, of the sequence SEQ ID NO: 1is thus revealed in fraction 15. Another peptide predominantly presentin fraction 21 also has a sequence of 23 amino acids which is identicalto that, for the N-terminal part, of the sequence SEQ ID NO: 2.

These results therefore demonstrate that the strain CNCM I-1351 producestwo peptides having an antibacterial activity. However, the differentappearance of the inhibition halos obtained between fractions 15 and 21,makes it possible to suspect a different antibacterial activity betweenthermophilin 1 and thermophilin 2 of the present invention.

On the other hand, the amino acid composition of fractions 15 and 21,previously hydrolysed with 6N HCl, at 100° C. for 24 h, is analysed bythe known method of "dabsyl chloride derivatization". The results showthat the amino acid composition of each fraction already appears tocorrespond to their respective peptide sequence.

Finally, fractions 15 and 21 are also subjected to mass spectometry, anda molecular weight which is of the order of 5800 Dalton is revealed forthermophilin 1, and a molecular weight which is of the order of 3900Dalton is revealed for thermophilin 2.

Sequencing of the genes for the bacteriocins

The degenerate nucleic sequences SEQ ID NO: 6 and SEQ ID NO: 7 describedin the sequence listing below, which correspond respectively to theN-terminal part and the C-terminal part of the thermophilin 1 peptidesequenced previously, are manufactured in a conventional manner.

A portion of the mixture of SEQ ID NO: 6 sequences is then renderedradioactive by the action of T4 polynucleotide kinase as described inthe laboratory manual "Molecular cloning, a laboratory manual" (secondedition, Sambrook et al., Cold Spring Harbor, Laboratory Press, 1989),called in the present disclosure "Maniatis".

PCR ("polymerase chain reaction") is then carried out with the aid ofthe two non-radioactive mixtures of the degenerate sequences SEQ ID NO:6 and SEQ ID NO: 7, on a chromosomal DNA preparation from the strainCNCM I-1351, as described in the manual "PCR techology" (H. A. Erdlicheditor, M stockton press, London).

A band of 128 base pairs (pb) is then revealed on an electrophoresisgel, which is then eluted according to Maniatis. A portion is thencloned directly into the plasmid pGEM-T (Promega) following therecommendations of the supplier, and is then sequenced by the"dideoxynucleotide" method, according to Maniatis, using the universalpUC19 probes. A probe having the sequence SEQ-ID NO: 8 described in thesequence listing below, corresponding to a sequence encoding amino acids9 to 47 of thermophilin 1, is thus obtained. Finally, the other portionof the eluted band of 128 pb is rendered radioactive by the methodcalled "random priming" according to Maniatis.

On the other hand, a digestion of a chromosomal DNA preparation from thestrain CNCM I-1351 is carried out with EcoRI and HindIII following therecommendations of the enzyme supplier, 10 μg of digestion product arethen run on an analytical electrophoresis gel, the DNA is transferred inalkaline medium from the gel onto a "Zeta probe" membrane (Biorad), themembrane is prehybridized at 54° C. overnight in a medium comprising 6×SSC, 1% SDS and 1% skimmed milk, then this membrane is hybridized to theradioactive degenerate probe SEQ ID NO: 6 in the previous hybridizationmedium, first for 18 h at 54° C., decreasing the temperature by 2° C.every 3 h, then for 24 h at 42° C. The membrane is then washed for 2min, three times in succession, in 6× SSC at room temperature, and for 1min in 6× SSC at 47° C. The membrane is finally exposed to anautoradiography film. All these steps are carried out according to theManiatis manual.

A 3.6 kb band is then revealed, which makes it possible for us to locatein a preparative electrophoresis gel of the chromosomal DNA (300 μg) ofthe strain CNCM I-1351 performed under the same conditions as describedabove, the gel portion comprising the piece of DNA which is of interest.This gel portion is then cut out and eluted in a conventional manner,and the eluted DNA is ligated to the vector pUC19 (Messing et al.,Methods Enzymol., 101:20, 1983) previously hydrolysed with EcoRI andHindIII. These steps are carried out according to the Maniatis manual.

The strain BZ234 of Escherichia coli (Biocentre collection, Universityof Bale, Switzerland) rendered competent beforehand, is thenconventionally transformed with the ligation medium. The transformedcells are then selected by α-complementation. Then according to themethod called "colony lift", according to Maniatis, 300 transformedcolonies are transferred to a filter, they are lysed, they arehybridized to the radioactive sequence SEQ ID NO: 8, then the filter isexposed to an autoradiography film.

13 colonies having a plasmid capable of hybridizing with the sequenceSEQ ID NO: 8 are then observed on the film. Two of these colonies arethen selected, the plasmid DNA is conventionally extracted therefrom,and the DNA fragment cloned into the two selected pUC19 plasmids issequenced by the "dideoxynucleotide" method, with the aid of universalpUC19 probes, then probes based on the sequences thus obtained.

A nucleic sequence SEQ ID NO: 3 described in the sequence listing belowis thus obtained which is identical for the two plasmids selected. Thissequence thus comprises an operon encoding two proteins having the aminoacid sequences SEQ ID NO: 4 corresponding before maturation tothermophilin 1, and SEQ ID NO: 5 corresponding before maturation tothermophilin 2 (see the sequence listing below). A third open readingframe also starts from nucleotide 679 of this sequence, and shouldcertainly correspond to the gene for immunity.

By comparing the N-terminal peptide sequences of the purifiedbacteriocins and the amino acid sequences of the proteins encoded by thecoding frames of the operon SEQ ID NO: 3, it can be determined that theprotein of amino acid sequence SEQ ID NO: 4 (thermophilin 1) has aleader peptide of 23 amino acids which has a Glycine-Glycine unitcharacteristic of a class of bacteriocins from lactic acid bacteria.Finally, the molecular mass of thermophilin 1, calculated from itsnucleic sequence, corresponds to that found by spectrometry, that is tosay is of the order of 5800 Dalton.

Likewise, the protein of amino acid sequence SEQ ID NO: 5 (thermophilin2) has a leader peptide of 21 amino acids, which has a Glycine-Glycineunit characteristic of a class of bacteriocins from lactic acidbacteria. Finally, the molecular mass of thermophilin 2, calculated fromits nucleic sequence, corresponds to that found by spectrometry, that isto say is of the order of 3900 Dalton.

Role of the different bacteriocins

A homology with the first peptide of the "lactococein M" operon(Klaenhammer et al., FEMS Micro. Rew., 12, 39-86, 1993) was found forthe sequence of thermophilin 1. This homology relates to the repetitionof a GA unit. Likewise, a homology with a gene for the "lactacin F"operon (Klaenhammer et al., cited above) was found for thermophilin 2,in the GenEMBL data bank using the TFASTA program from GCG.

The two lactococein M and lactacin F operons in fact encode porationcomplexes involving several peptides. It is therefore possible that theoperon previously described can encode peptides acting conjointly in aporation complex.

Nevertheless, it is not excluded that the two bacteriocins actindependently, because of the somewhat different inhibition haloobserved between the two thermophilins in the agar well test previouslydescribed.

EXAMPLES

The examples below are presented as illustration of the process ofproduction and of the uses of the bacteriocin according to the presentinvention. The percentages are given therein by weight unless wherestated otherwise.

Example 1

An M17 culture medium to which 1% sucrose has been added is inoculatedwith 1% (v/v) of a culture conaining 10⁸ microorganisms of the strainCNCM I-1351 of S. thermophilus per ml. The incubation is carried out for6 h at 42° C. under anaerobic conditions after which the medium containsabout 10⁸ microorganisms of the strain per ml and has an OD₆₀₀ of 3.6.

The standard culture thus obtained is centrifuged. The supernatant(standard) is collected. It is acidified to pH 1.5 with H₃ PO₄, aprecipitate is obtained which is removed by centrifugation and an acidicprecipitation supernatant is collected.

The bacteriocins contained in the latter are precipitated with 10%trichloroacetic acid. The precipitated bacteriocins are collected andthereafter resuspended in aqueous suspension with 0.2% trifluoroaceticacid (v/v).

The bacteriocins are reprecipitated with 10% trichloroacetic acid. Theprecipitated bacteriocins are collected, they are washed with 100%acetone and they are resuspended in aqueous suspension with 0.2%trifluoroacetic acid (v/v).

A standard crude extract of the present bacteriocins is obtained havingan activity of 1.410⁵ au/ml.

Table VI below gives some details on the characteristics of one litre ofstandard supernatant and on those of the 18 ml of standard crudeextract, in other words of concentrate which was obtained therefrom,especially in terms of protein content and antibacterial activity.

                                      TABLE V                                     __________________________________________________________________________                Total protein    au/mg                                                                              Total                                              Volume                                                                             (PIERCE kit)                                                                              au/mg                                                                              dry  activity                                           (ml) (mg)   au/ml                                                                              protein                                                                            weight                                                                             (au)                                        __________________________________________________________________________    Supernatant                                                                          1000 6700   4.6 × 10.sup.2                                                               68   --   4.6 × 10.sup.3                        Crude extract                                                                        18   83     1.4 × 10.sup.5                                                               2.5 × 10.sup.5                                                               1.4 × 10.sup.5                                                               2.1 × 10.sup.6                        __________________________________________________________________________

Example 2

Set-style yogurts are prepared comprising the strain of the invention S.thermophilus CNCM I-1351, and the strains ST11 of S. thermophilus (whichis resistant to the bacteriocins according to the invention butexhibiting no antibacterial activity) and YL5 of L. bulgaricus mentionedabove.

A milk based on whole milk comprising 3.7% fat and 2.5% skimmed milkpowder is thus prepared. 40 l of this milk are pasteurized at 92° C. for6 min, it is then homogenized at 75° C. and 150 bar (two stages),finally it is cooled to a temperature of about 42° C.

The freeze-dried strains S. thermophilus CNCM I-1351, S. thermophilusST11 and L. bulgaricus YL5 are then reactivated by several successiveprecultures in a sterile MSK medium (10% reconstituted powdered skimmedmilk comprising 0.1% of a commercial yeast extract).

The sterile milk is then inoculated in an amount of 1% (v/v) of thethird preculture of each S. thermophilus strain taken at the stage forcoagulation of the medium, and in an amount of 2% (v/v) of the thirdpreculture of the L. bulgaricus strain taken at the stage forcoagulation of the medium. The milk is then incubated at 42° C. up to apH of about 4.65, then it is cooled to 4° C.

For comparison, a traditional set-style yogurts is prepared in the samemanner as described above, with the previously described strains YS8 andSFi3 of S. thermophilus, and the strain YL18 of L. bulgaricus, which aretraditionally used for the manufacture of yoghurt.

Table VI below illustrates the characteristics of the products obtained,especially their pH during their preservation at 4° C.

                  TABLE VI                                                        ______________________________________                                                Time for   pH of the product                                                                          pH of the product                                     acidification                                                                            after 1 day  after 24 days                                 Examples                                                                              up to pH 4.65                                                                            (at 4° C.)                                                                          (at 4° C.)                             ______________________________________                                        Example 2                                                                             8 h 30     4.6          4.6                                           Comparative                                                                           6 h        4.34         4.3                                           example                                                                       ______________________________________                                    

Example 3

Mozzarella cheese is prepared in a traditional manner with the aid of anS. thermophilus CNCM-1351 culture.

Example 4

10 litres of a culture of the strain CNCM I-1351 of S. thermophilus areproduced in an M17 medium supplemented with 1% sucrose, for 6 h, at 42°C. and under anaerobic conditions. 200 g of XAD-7 resin (Sigma) are thenadded directly to the culture, the whole is stirred gently for 1 h at 4°C. The mixture is then filtered through a Schleicher & Schuell filter(Germany) No. 604, then the resin retained on the filter is washed with10 litres of a 50 mM acetic acid solution, pH 5.2, in order to removethe bacteria. 450 ml of a solution comprising 100% ethanol and 20 mMammonium acetate are then added to the resin, the whole is filtered inorder to remove the resin, then the filtrate is freeze-dried until apowder comprising the bacteriocins according to the invention isobtained which can be used in the food industry.

The antibacterial activity of this powder, previously diluted in water,is then determined by the agar well test described above. This powderexhibits 10⁷ au/g of powder.

Finally, 0.5 g/kg of the above powder is added to a meat mousse duringits preparation in a traditional manner. A meat mousse is thus obtainedcomprising 5.10³ au/g of bacteriocins capable of completely inhibitingthe development of pathogenic bacteria, especially Clostridium.

Example 5

This example relates to the preparation of a moisturizing cream for skincare containing 0.05 g/kg of the powder described in Example 4, that isto say therefore 510² au/g of bacteriocins capable of inhibiting thedevelopment of undesirable bacteria on the skin.

To manufacture this emulsion, the components of the lipid phase A aremixed and it is heated to 75° C. The aqueous phase B is prepared and itis also heated to 75° C., then it is added to the lipid phase A whilemixing slowly, and the mixture is then cooled, with slow mixing, to roomtemperature, that is to say about 25° C. At this temperature, theconstituents C are slowly added in the order of the formula.

    ______________________________________                                        Lipid Phase A                                                                                              %                                                ______________________________________                                        Peg-6-stearate, glycerate and peg-20-cethyl ether                                                          15                                               (peg:polyethylene glycol)                                                     Vaseline oil                 5                                                Wheat germ oil stabilized with 0.1% phenylindans (antioxidant)                                             3                                                and 1% soya bean phospholipids (see EP94109355.1)                             Sweet almond oils            2                                                Cetyl alcohol                1                                                Isostearyl isostearate       2                                                2-Octyl-dodecyl-myristate    1                                                Lanolin wax                  1                                                ______________________________________                                    

    ______________________________________                                        Aqueous Phase B                                                                                  %                                                          ______________________________________                                        Methylisothiazoline                                                                              0.1                                                        Demineralized water                                                                              59.6                                                       Human placenta protein                                                                           2                                                          ______________________________________                                    

    ______________________________________                                        Additives C                                                                                                  %                                              ______________________________________                                        Propylene glycol and calendula extract                                                                       2                                              50% soluble collagen in demineralized water                                                                  5.8                                            Perfume                        0.3                                            2.5% bacteriocin powder according to Ex. 4 in demineralized                                                  0.2er                                          ______________________________________                                    

Example 6

0.5 g/kg of the bacteriocin powder described in Example 4 is added to aliquid dentifrice. This dentifrice is thus capable of inhibiting thedevelopment of pathogenic bacteria of the buccal cavity, and especiallyStreptococcus sobrinus.

Example 7

A solution comprising the bacteriocin powder of Example 4 diluted inwater in an amount of 1%, is sprayed on a food product intended to besterilized in order to prevent post-contamination during packaging.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 8                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 62 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus thermophilus                                      (B) STRAIN: CNCM I-1351                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TyrSerGlyLysAspCysLeuLysAspMetGlyGlyTyrAlaLeuAla                              151015                                                                        GlyAlaGlySerGlyAlaLeuTrpGlyAlaProAlaGlyGlyValGly                              202530                                                                        AlaLeuProGlyAlaPheValGlyAlaHisValGlyAlaIleAlaGly                              354045                                                                        GlyPheAlaCysMetGlyGlyMetIleGlyAsnLysPheAsn                                    505560                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus thermophilus                                      (B) STRAIN: CNCM I-1351                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GlnIleAsnTrpGlySerValValGlyHisCysIleGlyGlyAlaIle                              151015                                                                        IleGlyGlyAlaPheSerGlyGlyAlaAlaAlaGlyValGlyCysLeu                              202530                                                                        ValGlySerGlyLysAlaIleIleAsnGlyLeu                                             3540                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 770 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus thermophilus                                      (B) STRAIN: CNCM I-1351                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 221..475                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: 221..289                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 290..475                                                        (D) OTHER INFORMATION: /function= "encodes for                                thermophiline 1"                                                              (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 495..686                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: 495..557                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 558..686                                                        (D) OTHER INFORMATION: /function= "encodes for                                thermophiline 2"                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AATGGCACGAACGTCCTGAATGGTTAAAAGATATTTCGGATCTTCCTAAAAAATACATAC60                TGAACGGTCGCTTTCCCTTCTTGAATGGTAAAATTTTCCCATTAGGAAAGTTAAATGACT120               GTTCAAGAAATGGGGAAATTATTTTTTGAAGTAGTGCTATACTAGACTTGTCAAGGTTGC180               AACCCGACAAAATAAAAATATTAGGTAGGAGATATTTACAATGAATACAATAACT235                    MetAsnThrIleThr                                                               23-20                                                                         ATTTGTAAATTTGATGTTTTAGATGCTGAACTTCTTTCGACAGTTGAG283                           IleCysLysPheAspValLeuAspAlaGluLeuLeuSerThrValGlu                              15-10-5                                                                       GGTGGATACTCTGGTAAGGATTGTTTAAAAGACATGGGAGGATATGCA331                           GlyGlyTyrSerGlyLysAspCysLeuLysAspMetGlyGlyTyrAla                              1510                                                                          TTGGCAGGAGCTGGAAGTGGAGCTCTGTGGGGAGCTCCAGCAGGAGGT379                           LeuAlaGlyAlaGlySerGlyAlaLeuTrpGlyAlaProAlaGlyGly                              15202530                                                                      GTTGGAGCACTTCCAGGTGCATTTGTCGGAGCTCATGTTGGGGCAATT427                           ValGlyAlaLeuProGlyAlaPheValGlyAlaHisValGlyAlaIle                              354045                                                                        GCAGGAGGCTTTGCATGTATGGGTGGAATGATTGGTAATAAGTTTAAC475                           AlaGlyGlyPheAlaCysMetGlyGlyMetIleGlyAsnLysPheAsn                              505560                                                                        TAAGGAAGGAGTTTATATCATGAAGCAGTATAATGGTTTTGAGGTTCTACAT527                       MetLysGlnTyrAsnGlyPheGluValLeuHis                                             21- 20-15                                                                     GAACTTGACTTAGCAAATGTAACTGGCGGTCAAATTAATTGGGGATCA575                           GluLeuAspLeuAlaAsnValThrGlyGlyGlnIleAsnTrpGlySer                              10-515                                                                        GTTGTAGGACACTGTATAGGTGGAGCTATTATCGGAGGTGCATTTTCA623                           ValValGlyHisCysIleGlyGlyAlaIleIleGlyGlyAlaPheSer                              101520                                                                        GGAGGTGCAGCGGCTGGAGTAGGATGCCTTGTTGGGAGCGGAAAGGCA671                           GlyGlyAlaAlaAlaGlyValGlyCysLeuValGlySerGlyLysAla                              253035                                                                        ATCATAAATGGATTATAAAAGTCTTTTATCGCTTTTATTATTCATAATTCCCCTT726                    IleIleAsnGlyLeu                                                               40                                                                            GTAGTTATACTAATCGTTCTTCGAAAGAATAATCAGAAACTAAT770                               (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 85 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetAsnThrIleThrIleCysLysPheAspValLeuAspAlaGluLeu                              23-20-15- 10                                                                  LeuSerThrValGluGlyGlyTyrSerGlyLysAspCysLeuLysAsp                              515                                                                           MetGlyGlyTyrAlaLeuAlaGlyAlaGlySerGlyAlaLeuTrpGly                              10152025                                                                      AlaProAlaGlyGlyValGlyAlaLeuProGlyAlaPheValGlyAla                              303540                                                                        HisValGlyAlaIleAlaGlyGlyPheAlaCysMetGlyGlyMetIle                              455055                                                                        GlyAsnLysPheAsn                                                               60                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 64 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       MetLysGlnTyrAsnGlyPheGluValLeuHisGluLeuAspLeuAla                              21-20-15-10                                                                   AsnValThrGlyGlyGlnIleAsnTrpGlySerValValGlyHisCys                              51510                                                                         IleGlyGlyAlaIleIleGlyGlyAlaPheSerGlyGlyAlaAlaAla                              152025                                                                        GlyValGlyCysLeuValGlySerGlyLysAlaIleIleAsnGlyLeu                              303540                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GAYATGGGNGGNTAYGC17                                                           (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GCTATNGCNCCNACGTG17                                                           (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 128 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus thermophilus                                      (B) STRAIN: CNCM I-1351                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GATTGTTTAAAAGACATGGGAGGATATGCATTGGCAGGAGCTGGAAGTGGAGCTCTGTGG60                GGAGCTCCAGCAGGAGGTGTTGGAGCACTTCCAGGTGCATTTGTCGGAGCTCATGTTGGG120               GCAATTGC128                                                                   __________________________________________________________________________

What is claimed is:
 1. An isolated streptococcus thermophilusbacteriocin having the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2.2. A process for preparing a food or cosmetic product which comprisesadding at least one bacteriocin according to claim 1 to the product inan amount effective to impart antibacterial activity to the product. 3.The process of claim 2 wherein the bacteriocin is added in the form ofan extract which is obtained by culturing a Streptococcus thermophilousstrain in a medium under conditions favorable to the growth of thestrain to form a culture which contains about 10⁷ to 10⁹ microorganismsof the strain per ml, centrifuging the culture to produce a supernatantwhich contains the bacteriocin, and obtaining the extract from thesupernatant.
 4. The process of claim 2 wherein the bacteriocin is addedby incorporating a Streptococcus thermophilous strain in the product. 5.The process of claim 4 wherein a culture of the Streptococcusthermophilous strain is used as a starter in the preparation of a cheeseor acidified milk product.
 6. The process of claim 4 wherein the strainis CNCM I-1351.
 7. The process of claim 2 wherein the product is a meatproduct, a cream, a lotion or an oral health product and the bacteriocinis present in the product in an amount effective against pathogenicbacteria.